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Vaccine 25 (2007) 6313–6320

Robust ��+ T cell expansion in infants immunized atbirth with BCG vaccine�

T.N. Mazzola a,b,1, M.T.N. Da Silva a,b,2, Y.M.F. Moreno a,b,1, S.C.B.S. Lima a,b,1,E.F. Carniel a,b,3, A.M. Morcillo a,b,3, M.A.R.G.M. Antonio a,b,3, M.L. Zanolli a,b,3,

A. Aranha Netto a,b,3, M.H. Blotta b,c,3, I. Raw d,4, M.M.S. Vilela a,b,∗a Center of Investigation in Pediatrics, Pediatrics Department, State University of Campinas Medical School, Campinas, Sao Paulo, Brazil

b Rua Tessalia Vieira de Camargo, 126, Campinas, Sao Paulo, CEP 13083-887, Brazilc Department of Clinical Pathology, State University of Campinas Medical School, Campinas, Sao Paulo, Brazil

d Instituto Butantan, Rua Vital Brasil, 1500, Sao Paulo, Sao Paulo, CEP 05503-900, Brazil

Received 3 April 2007; received in revised form 15 June 2007; accepted 15 June 2007

bstract

Cell-mediated immune responses to BCG vaccine were evaluated in 7-month-old infants vaccinated with intradermal combined BCG andepatitis B or intradermal BCG and intramuscular Hepatitis B at birth. Peripheral blood mononuclear cell cultures from both groups showedD4+, CD8+ and remarkable ��+ T cell BCG-specific proliferation, without significant differences. Also, IL-10, IL-12, IFN-� and TNF-�

oncentrations in culture supernatants, measured by ELISA, were similar. The results suggested that the combined BCG and Hepatitis Baccine was as immunogenic as BCG separated from Hepatitis B vaccine.

2007 Elsevier Ltd. All rights reserved.

eywords: BCG vaccine; Cellular immunity; Combined vaccines

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abprotect against severe forms of systemic tuberculosis (TB) in

� Preliminary results of these findings were presented at the NewApproaches to Vaccine Development—From bench to the field, Berlin,Germany, 8–10 September 2005.

∗ Corresponding author. Tel.: +55 19 35218963; fax: +55 19 35218972.E-mail addresses: tanmazzola@yahoo.com.br (T.N. Mazzola),

nolasco@fcm.unicamp.br (M.T.N. Da Silva), yarafm@fcm.unicamp.br(Y.M.F. Moreno), scbsl@uol.com.br (S.C.B.S. Lima),carniel@fcm.unicamp.br (E.F. Carniel), morcillo@fcm.unicamp.br(A.M. Morcillo), anze@fcm.unicamp.br (M.A.R.G.M. Antonio),zanolli@fcm.unicamp.br (M.L. Zanolli), abimael@fcm.unicamp.br(A.A. Netto), heblotta@fcm.unicamp.br (M.H. Blotta),iraw@butantan.gov.br (I. Raw), marlucevilela@terra.com.br(M.M.S. Vilela).

1 Tel.: +55 19 35218989; fax: +55 19 35218972.2 Tel.: +55 19 35218979; fax: +55 19 35218972.3 Tel.: +55 19 35218973; fax: +55 19 35218972.4 Tel.: +55 11 37262139; fax: +55 11 37261505.

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264-410X/$ – see front matter © 2007 Elsevier Ltd. All rights reserved.oi:10.1016/j.vaccine.2007.06.039

. Introduction

With the increasing number of recommended children’saccines it is often required that an infant receive multiplenjections, which is disconcerting to parents and health careroviders [1–3]. The value of combined vaccines relies onbtaining high levels of age-appropriate immunization, theossibility of reducing the number of underimmunized chil-ren, and in simplifying immunization schedules. There isneed, therefore, to demonstrate the safety and efficacy of

ombined vaccines by clinical trials [4–6].Despite the controversy concerning its efficacy against

dult pulmonary tuberculosis, intradermal Mycobacteriumovis Bacillus Calmette-Guerin (BCG) vaccine can, at least,

hildren, particularly meningitis and miliary disease, and soorld Health Organization has encouraged vaccination [7].oreover, there is no known standardization of protective

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314 T.N. Mazzola et al. / V

mmunity; neither the post-vaccination scar nor the tuber-ulin tests are recognized as protection markers [8]. A newaccine against Mycobacterium tuberculosis is needed but its still necessary to find out which immune response can leado protection and thus reach this goal [7].

Regarding the evaluation of BCG immunogenicity, Cour-aget et al. [9] used tuberculin reactivity as a surrogatearker, and Ota et al. [10] evaluated BCG immunogenicityith a radioactive proliferation assay using PPD as anti-en. In our study, BCG immunogenicity was measured withflow cytometry-based proliferation technique that allows

he determination of expanding lymphocyte subsets, and thencubation with viable bacilli allows for a more detailedxploration of the complexity of antigen presentation androcessing.

In children, there is no experience of an intradermal BCGombined vaccine. Since young children are very suscepti-le to systemic tuberculosis and there are only a few studiesf BCG-responses in childhood [11], in this article cellularmmune responses to BCG in vitro from 7-month-old infantsmmunized at birth with a single dose of combined BCG withecombinant Hepatitis B surface antigen (HBsAg) vaccine areescribed.

. Materials and methods

.1. Study participants and vaccination

This cross-secctional study was conducted between Octo-er 2004 and December 2005. Study subjects were selected assample from a population of 498 children who took part inphase III randomized controlled trial. Infants were immu-ized at birth either with combined intradermal BCG andepatitis B or intradermal BCG and intramuscular Hepatitisvaccine into the antero-lateral area of the thigh. All vaccinesere provided by Instituto Butantan, Sao Paulo, SP, Brazil.total of 85 healthy newborns were selected. The preterm

gestational age ≤37 weeks) and the low birth weight new-orns (weight <2500 g), infants with congenital or geneticefects or whose mothers were younger than 18-years-oldr had Hepatitis B carrier status, infants with family historyf TB, Syphilis or Human Immunodeficiency Virus (HIV)nfection were excluded. The type of vaccine administeredas revealed to the entire research team only at the prepa-

ation of this report, but vaccinators and vaccine recipientsad knowledge of which vaccine was being injected since theeginning of the study.

Infants from one group had received combined intrader-al BCG and Hepatitis B, which was prepared as follows:solution of purified recombinant HBsAG protein (100 �g)

nd BCG Moreau Rio de Janeiro strain suspension (1 mg)

ere mixed in 4 mL vials containing 2% sodium glutamate

s stabilizer and lyophilized. Immediately before adminis-ration the vaccine was reconstituted in 1 mL of 0.85% NaClolution, without preservative. The combined vaccine was

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5 (2007) 6313–6320

vailable in amber glass vials with 10 lyophilized doses,.1 mL each, containing 0.1 mg of BCG and 10 �g of HBsAg.

Children from the other group had received intradermalCG (0.1 mg lyophilized BCG Moreau strain in 0.85% NaClith 1.1 mg sodium glutamate), stored in amber glass vialsith 10 doses of 0.1 mL each, and intramuscular Hepatitis Baccine (BUTANG®, 10 �g HbsAg with 0.625 mg aluminumydroxide and 0.05 mg thimerosal), available in glass vials of0-dose liquid solution, 0.5 mL each. Both groups receivedhe second and third doses of intramuscular Hepatitis B vac-ine (BUTANG®) at 1 and 6 months of age, respectively. Thetudy protocol was approved by the Research Ethics Com-ittee from the State University of Campinas (UNICAMP),ampinas, SP, Brazil. Written informed consent was obtained

rom each infant’s parent or legal guardian.

.2. Blood collection

Ten milliliters of heparinized peripheral blood were col-ected and used to evaluate immune responses to BCGaccination at 7 months of age. For ethical reasons, it was notossible to access immune response from children at samege not vaccinated with BCG. So, 10 cord blood samplesrom healthy newborns were used as negative controls forCG vaccination.

.3. Antigens used in cell culture assays

Lyophilized BCG Moreau Rio de Janeiro vaccine vialsere freshly reconstituted with RPMI 1640 (Sigma, USA)

nd used at 5 × 105 UFC/mL. Phytohemagglutinin (PHA,igma, USA) was used as a positive control at 7.5 �g/mL.

.4. BCG-specific T cell proliferation

The protocol for proliferation assay was carried out onresh blood, and was adapted from Gaines et al. [12]. Periph-ral blood mononuclear cells (PBMC) from infants and cordlood mononuclear cells (CBMC) were isolated by den-ity gradient centrifugation over Ficoll-Hypaque (Amershamiosciences, USA), washed, diluted to 1 × 106 cells/mL inPMI 1640 medium (Sigma, USA) supplemented with 10%uman AB serum (Sigma, USA), 1% glutamine (Sigma,SA) and 0.1% gentamycin and stimulated for 6 days with

econstituted BCG, PHA or medium alone at 37 ◦C with% CO2 in round-bottomed 96-well tissue culture platesNUNC, Denmark). After harvesting with 20 mM ethyleneiamine tetracetic acid (EDTA), samples were incubated withuman immunoglobulin and then stained with anti-humanD3, CD4, CD8 and T cell receptor (TCR) pan �� (Beck-an Coulter, USA or France or Caltag Laboratories, USA)uorescent antibodies before acquisition (Epics XL-MCL

ow cytometer, Beckman-Coulter, USA) and analysis (Expo,SA). Isotype controls were used to discriminate positiveopulations (Beckman Coulter, USA). Only CD3+ T cellsere used in analysis. Forward and side scatters were used

T.N. Mazzola et al. / Vaccine 25 (2007) 6313–6320 6315

Fig. 1. Flow cytometry of unstimulated (control), Bacillus Calmette-Guerin (BCG) and phytohemagglutinin (PHA)-stimulated peripheral blood mononuclearc at birth( ed (B).l

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ells of a 7-month-old infant vaccinated with combined BCG and Hepatitis BFS and SS gates), from which resting and blast lymphocytes were separatymphocytes (C and D).

o gate on resting and blast lymphocytes. Dead cells werexcluded from all analyses. CD4+, CD8+ and TCR ��+ cellsere identified in the gate of blast lymphocytes. Prolifera-

ion was measured by CD3+ percent blasts, in which baselineroliferation was subtracted from BCG and PHA-stimulatedultures (Fig. 1).

.5. BCG-specific cytokine production in vitro

PBMC and CBMC were diluted to 2 × 106 cells/mLn supplemented RPMI and incubated for 48 h in round-ottomed 96-well tissue culture plates with BCG, PHA

r medium alone at 37 ◦C. Supernatants were collectednd stored at −80 ◦C. Interferon-� (IFN-�), tumor necro-is factor-� (TNF-�), interleukin 10 (IL-10) and IL-12 levelsere determined in duplicate by a two-monoclonal Antibody

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. After gating on CD3+ cells (A), events are analyzed for size and complexityT lymphocyte subsets (CD4+, CD8+ and TCR ��+) were verified in blast

andwich Enzyme Linked Immunosorbent Assay (two-MAbLISA) (R&D Systems, USA for IFN-�, TNF-� and IL-10nd Amersham Biosciences, UK for p40 and p70 IL-12) inat-bottomed MultiSorp ELISA plates (NUNC, Denmark),ccording to the manufacturers’ protocols. IL-12 concentra-ion was not performed in cord blood samples. Recombinantytokine was used for the standard curve. The limit of detec-ion was 15.6 �g/mL for IFN-� and TNF-�, 46.9 �g/mL forL-10 and 5.0 �g/mL for IL-12.

.6. Statistical considerations

All analyses were done with SPSS® for Windows (Version.5.1, USA). Data analysis was performed with χ2, Friedman,ann–Whitney, nonparametric multiple comparison and

pearman correlation tests (p < 0.05). All reported p-values

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Fig. 3. Distribution of CD3+ percent blasts determined by flow cytometryof Bacillus Calmette-Guerin (BCG: (�)) and phytohemagglutinin (PHA:(�))-stimulated peripheral blood mononuclear cells from infants vaccinatedwith BCG and Hepatitis B vaccine at birth or cord blood mononuclear cells.Medians (indicated by bars) of CD3+ blasts in BCG-stimulated cultures were22.8 and 10.4% for vaccines and cord blood, respectively. Medians of CD3+

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316 T.N. Mazzola et al. / V

re two-tailed. GraphPad Prism (Version 4.0, GraphPad Soft-are, USA) was employed to perform the proliferation and

ytokine figures.

. Results

.1. Study participants

Forty-seven infants (24 male and 23 female) in the com-ined vaccine group and 38 (17 male and 21 female) inhe separate vaccine group were studied for BCG immuneesponse. The combined vaccine group median age was.34 months (range 6.3–8.1) and 7.38 months in separateaccine group (range 7.0–8.7). There were no differencesn terms of gender (χ2 = 0.337, d.f. = 1, p = 0.562) nor ageMann–Whitney U = 872.5, p = 0.856) between the two vac-ination groups. No local or systemic adverse reactions werebserved after BCG vaccinations.

.2. BCG-specific T cell proliferation

T cell proliferation was measured by flow cytometryFig. 1). CD3, CD4 and CD8 immunophenotyping was car-ied out in the cultures of all the infants and TCR ��+ blastells were analyzed in 46 children (29 of the combined vac-ine group).

Median background CD3+ proliferation was 2.17% in theombined vaccine group and 2.26% for separate vaccine

roup. Percent blasts in cultures with live BCG (medians of0.39 and 28.20% for combined and separate vaccine groups,espectively, p = 0.185) and PHA (medians of 62.53 and0.52% for combined and separate vaccine groups, respec-

ig. 2. Distribution of CD3+ percent blasts determined by flow cytometryf Bacillus Calmette-Guerin (BCG: (�)) and phytohemagglutinin (PHA:�))-stimulated peripheral blood mononuclear cells from combined or sep-rated BCG and Hepatitis B vaccinated infants. Medians (indicated byars) of CD3+ blasts in BCG-stimulated cultures were 20.4 and 28.2% forombined and separated vaccines, respectively. Medians of CD3+ blasts inHA-stimulated cultures were 62.5 and 60.5% for combined and separatedaccines, respectively; n: number of individuals.

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lasts in PHA-stimulated cultures were 61.7 and 57.7% for vaccines and cordlood, respectively. Mann–Whitney test was used to verify significances; n:umber of individuals.

ively, p = 0.965) did not differ between groups (Fig. 2). Forhis reason, we compared all of them to cord blood prolifer-tion.

In the cord blood samples median background prolifer-tion was 13.02% and CD3+ percent blasts in cultures withHA were similar to vaccinated infants (medians of 61.71 and7.73% for infants and cord blood, respectively, p = 0.265).here was a difference between proliferation in cultures of

nfants and cord blood with live BCG (medians of 22.84 and0.37% for infants and cord blood, respectively, p = 0.006).he comparisons are illustrated in Fig. 3.

The frequency of BCG- and PHA-proliferating CD4+,D8+ and ��+ cells were also similar between vaccinationroups (Table 1). When we compared the frequency of CD4+,

able 1ercentages of CD4+, CD8+ and ��+ effector cells determined by flowytometry in PBMCa cultures incubated with live BCG or PHA from 7-onth-old infants vaccinated with combined or separated BCG and Hepatitisvaccines

timuli T cell Median% of T cell (n)b p-value

Combined Separated

CGcCD4+ 32.6 (47) 27.35 (38) 0.052CD8+ 12.1 (47) 12.2 (38) 0.898TCR ��+ 58.3 (29) 70.3 (17) 0.078

HAdCD4+ 87.9 (47) 90.25 (38) 0.238CD8+ 9.1 (47) 8.15 (38) 0.269TCR ��+ 0.6 (29) 0.6 (17) 0.414

a Peripheral blood mononuclear cell.b Number of individuals.c Bacillus Calmette-Guerin.d Phytohemagglutinin.

T.N. Mazzola et al. / Vaccine 2

Fig. 4. Distribution of proliferating CD4+ (�), CD8+ (�) and ��+ (�) Tcells determined by flow cytometry of Bacillus Calmette-Guerin (BCG, A)and phytohemagglutinin (PHA, B) stimulated peripheral blood mononu-clear cells from infants vaccinated at birth with BCG. Values representpercents. Number of individuals, n = 46. Medians (indicated by bars) ofCD4+, CD8+ and ��+ blasts in BCG-stimulated cultures were 30.0, 12.5and 60.2%, respectively. Medians of CD4+, CD8+ and ��+ blasts in PHA-si(

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timulated cultures were 88.7, 9.0 and 0.6%, respectively. Different lettersndicate statistical significance by nonparametric multiple comparison testp < 0.05).

D8+ and ��+ cells in 46 vaccines (Fig. 4A and B) thereas a significantly difference between the T cell subpopula-

ions for control, BCG- and PHA-stimulated cultures, withigh percentages of ��+ cells in BCG cultures (Friedman< 0.001; nonparametric multiple comparison tests p < 0.05

n all cases).

.3. BCG-specific cytokine production

BCG-specific cytokines in cell cultures of infants andord blood were determined by ELISA after 48 h stimulationith BCG. The amount of cytokine secretion in supernatantsas compared among the groups of vaccines. Undetectabler small amounts of TNF-�, IFN-�, IL-10 and IL-12 werebserved in unstimulated samples (negative controls). IFN-, TNF-�, IL-10 and IL-12 production (Fig. 5) was notifferent in BCG-stimulated cultures of infants vaccinatedith combined or separated BCG and only IFN-� concen-

ration in control and PHA-stimulated cultures was differentetween the groups (p = 0.026). There was a positive correla-ion between IFN-� and TNF-� (p = 0.003), IFN-� and IL-12p = 0.007), TNF-� and IL-12 (p < 0.001) and IL-10 and IL-

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5 (2007) 6313–6320 6317

2 (p = 0.036). However, there was no correlation among theroliferation and cytokine assays.

Unstimulated samples of CBMC produced undetectabler small amounts of IFN-�, IL-10 and TNF-�. IFN-� pro-uction was also low in cord blood cultures with BCGnd PHA. Comparison of BCG-vaccinated infants and cordlood in vitro cytokine release (Fig. 6) showed differencen IFN-� and TNF-� concentrations of control and stimu-ated cultures (p < 0.05). On the other hand, IL-10 level wasimilar in cultures with BCG and PHA of infants and cordlood.

. Discussion

Our main goal in this study was to examine whether aCG and Hepatitis B combined vaccine could prime simi-

ar cell-mediated responses that BCG vaccine injected aloneoes. Comparable post-BCG vaccination cytokine and cellroliferation responses in 7-month-old vaccines were found.e also observed similar responses to Hepatitis B vaccine

data not shown). Given the requirement of a Th-1 typemmune response in protective immunity against mycobacte-ia, we reasoned that vaccine efficacy would be characterizedy antigen-specific lymphoproliferative responses and Th-1rofile cytokine production.

Our results showed that the combined BCG and Hepatitisvaccine was as immunogenic as BCG vaccine inocu-

ated alone, regardless of the assay used to evaluate theaccination response. Combining BCG with Hepatitis B inhe same injection could simplify immunization schedule,nd increase BCG vaccinal coverage if it is administeredn maternities. Additionally, intradermal immunization inhis way could result in the induction of strong effectormmune responses by the stimulation of newborn Langerhansells.

Simultaneous vaccination of BCG and Hepatitis B wastudied in the past. The results from Coursaget et al. [9]howed that intramuscular Hepatitis B vaccine given at birthoncurrently with BCG did not interfere with the devel-pment of the BCG vaccinal scars nor with the anti-HBseometric mean titres. BCG induced a Th1-type immuneesponse in infants at birth, with high in vitro concentra-ions of IFN-�, strong proliferative responses to PPD andow production of type-2 cytokines [10]. In addition, cytokineIFN-�, IL-5 and IL-13), proliferative and IgG responses toBsAg were significantly higher in infants vaccinated withCG and Hepatitis B at birth than in infants who had not

eceived BCG. This effect was already apparent at 2 monthsf age, after a single dose of Hepatitis B vaccine, and wasurther enhanced after the administration of the three dosesf HBsAg [10]. The current trial reinforces the results above,

nd additionally demonstrates specific ��+ T-cell lympho-yte expansion and a Th-1 pattern of cytokine production,ithout interference of HbsAg in cellular immune response

o BCG vaccine.

6318 T.N. Mazzola et al. / Vaccine 25 (2007) 6313–6320

Fig. 5. IFN-� (A), IL-10 (B), IL-12 (C) and TNF-� concentration (D) determined by ELISA (values are given in picograms per milliliter) by peripheral bloodm s Calmi cines ats

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ononuclear cells in cultures without stimuli (control: (�)) or with Bacillunfants immunized with separated or combined BCG and Hepatitis B vacignificances; n: number of individuals.

Our second goal was to describe in vitro cellular immu-ity to live BCG in vaccinated infants. The medians ofD4+, CD8+ and ��+ T cell BCG-stimulated proliferation

n the analysis of infants from both groups together were0.05, 12.50 and 60.20%, respectively. These T cell sub-opulations were shown to be significantly increased inive BCG-stimulated cultures of PBMC from adults vac-inated with BCG compared with nonvaccinated controls.owever, only ��+ T cells presented significant prolifera-

ion to M. tuberculosis when whole lysate was used as thetimulus [13]. Hussey et al. [14] observed that heat-killedCG-stimulated significantly greater proliferation, cytotoxicctivity and IFN-�, IL-10 and IL-5 production by PBMC from0-week-old infants vaccinated at birth with BCG than unvac-inated infants at same age. We demonstrated that infants atmonths of age showed significant TNF-�, IFN-�, IL-12

nd IL-10 production by BCG-stimulated PBMC in com-

arison to unstimulated cultures. Also, IL-10 was the onlyytokine studied in which production was similar betweenBMC and PBMC. This pattern toward a type-2 profile mighte attributed to the dominant type-2 cytokine milieu of preg-

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ette-Guerin (BCG: (�)) or phytohemagglutinin (PHA: (�)) stimulation inbirth. Bars indicate medians and Mann–Whitney test was used to verify

ancy and/or to immaturity of neonatal antigen-presentingells [15].

In humans and animal models the interaction betweenFN-� secreting CD4+ T cells and mycobacterial-infectedacrophages is known to be essential in the protective

esponse [16,17]. Neonatal BCG vaccination was associ-ted with increased IFN-� in vitro production in responseo live BCG, purified protein derivative of M. tubercu-osis (PPD) and other M. tuberculosis antigens [18,19].espite the immaturity of the immune system at birth,CG vaccination of neonates induced similar in vitro IFN-�roduction in response to PPD and live BCG in vacci-ated infants and adult controls [18,19]. Our results confirmhat PBMC from vaccinated infants showed CD4+ T cellroliferation and significant IFN-� release following BCGtimulation.

M. tuberculosis remains inside the phagosome and perfo-

ates the phagosomal membrane to reach cytosolic nutrients,timulating CD8+ T cells as mycobacterial antigens accesshe MHC class I pathway. CD8+ T cell perforin andranulysin-mediated mechanism participate in the lysis of

T.N. Mazzola et al. / Vaccine 2

Fig. 6. IFN-� (A), IL-10 (B) and TNF-� (C) production determined byELISA (values are given in picograms per milliliter) in cell cultures withoutstimuli (control: (�)) or with Bacillus Calmette-Guerin (BCG: (�)) or phy-tohemagglutinin (PHA: (�)) stimulation in peripheral blood mononuclearcells from BCG-vaccinated infants and cord blood mononuclear cells. Barsindicate medians and Mann–Whitney test was used to verify significances;n: number of individuals.

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nfected macrophages [17]. We observed CD8+ T cell pro-iferation stimulated by live BCG in vitro, although slighterhan CD4+ and ��+ T cell expansion. CD8 T-cell activation,erforin production and cytotoxic T-cell activity were greatlyeduced in TB patients when compared to healthy adults vac-inated with BCG at childhood [20]. In adults vaccinated withCG, PPD and live BCG proved capable of stimulating andctivating CD8+ T cells which produced IFN-� and TNF-�ut not IL-4. After stimulation with live BCG, CD8+ T cellsere positive for perforin and showed CTL activity againstCG [21].

We observed a remarkable expansion of ��+ T cells inesponse to BCG stimulation. Human ��+ T cells are stimu-ated by phospholigands, which are abundant in mycobacteria22]. Reduced �� and increased �� T cells counts werebserved in 2-month-old infants vaccinated with BCG at birth23]. In BCG-vaccinated adults, ��+ T cells proliferated afterive BCG stimulation in vitro, but not to the same dose of heat-illed BCG [13]. Our results pointed ��+ cells as the majorubset presenting expansion in cultures with live BCG evenfter 7 months of vaccination with BCG.

In mice ��+ T cells may regulate granuloma formation androtect against dissemination of the tuberculous challenge24]. Early after BCG vaccination of pigs, the proliferativeesponse of ��+ lymphocytes was higher than that of CD4+

cells [25]. Following vaccination with BCG, an increasen vitro natural killer cytolytic activity was observed in ��+

ymphocytes from pigs. Moreover, ��+ T cells respondedrior to cytotoxic CD8+ T cells to in vitro assays againstCG-infected monocytes [26]. It was shown that ��+ Tells from BCG-vaccinated animals expanded with heat-illed M. tuberculosis in the absence of CD4+ T cells butheir IFN-� production was dependent on CD4+ T celltimulation [25]. Recent data showed that activated human�+ T cells to become antigen-presenting cells capable of

nduction of CD4+ and CD8+ T cell response. Further-ore, �� T cells incubated previously with PPD inducedHC class II dependent CD4 T cell proliferation [27,28].

�+ T cells could, therefore, be responsible for mobiliz-ng the first line of innate and adaptive defenses againstuberculosis.

Antibody depletion of CD8+, NK1.1+ and ��+ cells inD4−/− mice that had been immunized with an attenuated. tuberculosis vaccine did not eliminate the pulmonary

rotective responses evoked by immunization, suggesting aole for other CD4, CD8 double-negative �� T cells in TBmmunity [24]. In our work, it is likely that the majorityf double-negative proliferating T cells were ��+ cells, bute cannot exclude the possible participation of other T cell

ubsets.Although the mechanisms that account for insufficientcell-dependent protection in response to BCG vaccina-

ion and M. tuberculosis infection remain unclear, studyingycobacteria-induced T cell subsets, especially ��+ T cells

nd cytokines, can contribute to the definition of the relevant

cell population for the design of new vaccines.

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Immunol Methods 2005;297(1–2):1–11.

320 T.N. Mazzola et al. / V

cknowledgements

We thank all the parents which consented their childrenarticipation in this clinical trial. We also are grateful to Dr.atherine Luzuriaga for critically reading this manuscript

nd offering useful suggestions. This project was supportedy grant number 2002/05666-2 Fundacao de Amparo aesquisa do Estado de Sao Paulo (FAPESP), Sao Paulo,razil.

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